Process for making membranes

ABSTRACT

A process for the preparation of a filtration membrane, which includes providing an aqueous suspension of vesicles having transmembrane proteins incorporated therein, the vesicles being formed from an amphiphilic block copolymer having reactive end groups; providing a porous support; functionalizing a surface of the porous support to introduce reactive groups on the surface which are capable of reacting with the reactive end groups of the amphiphilic block copolymers of the vesicles; depositing said suspension of vesicles on a surface of the porous support; and providing reaction conditions such that covalent bonds are formed between the vesicles and the surface.

PRIORITY CLAIM

This application is a continuation of U.S. patent application Ser. No.16/793,920, filed Feb. 18, 2020, which is a continuation of U.S. patentapplication Ser. No. 15/128,718, filed Sep. 23, 2016, now U.S. Pat. No.10,576,434, issued Mar. 3, 2020, which is a National Phase entry of PCTApplication No. PCT/EP2015/056292, filed Mar. 24, 2015, which claimspriority from Great Britain Patent Application Number 1405390.4, filedMar. 26, 2014, the disclosures of which are hereby incorporated byreference herein in their entirety.

FIELD OF THE INVENTION

The present invention relates to a process for making membranes.Specifically, it relates to a process for making water filtrationmembranes.

BACKGROUND OF THE INVENTION

Conventional nanofiltration or reverse osmosis water filtrationmembranes have been known for many decades. Typically, they are made bycasting a support membrane (often polysulfone or polyethersulfone);immersing the resulting cast in an aqueous solution of a diamine;removing excess from the surface of the membrane; immersing the membranein an organic solution of a trifunctional acyl halide; and curing theresulting product to produce a polyamide layer. Washing and secondarycoating are then carried out as necessary.

It is known from WO 01/32146 that membrane proteins may be incorporatedinto the walls of vesicles made from amphiphilic ABA block copolymers.This document includes extensive discussion of the nature of thepolymers, and discloses that the polymers may have polymerizable groupsat both chain ends. These polymerizable groups can be polymerized afterthe formation of the self-assembled vesicles, the polymerisationoccurring exclusively intravesicularly. WO 2004/011600 discloses thataquaporins may be incorporated into tri-block co-polymers to form amembrane which will only pass water, excluding all contaminants. Sincethese disclosures, much work has been carried out to developcommercially viable membranes incorporating transmembrane proteins, andparticularly water filtration membranes based on aquaporins. Thechallenge is to produce a working membrane, which is physicallysufficiently robust to withstand the necessary conditions.

WO 2009/076174 describes a method of preparing substantially flatmembranes based on block copolymers and aquaporins. Dong et al, J. Mem.Sci. 2012, 409-410, 34-43, creates block-copolymer vesiclesincorporating an aquaporin, but then breaks the vesicles using a vesiclerupturing method to deposit a planar monolayer of polymer on the surfaceof a support. WO 2010/146365 describes vesicles which may haveaquaporins embedded in them, suspended in an oil phase to form a liquidmembrane. According to Zhao et al, J. Membrane Sci. 2012, 422-428,various proposed methods of producing aquaporin membranes includepolymer tethered bio-layers, biomembrane aperture partition arrays,membrane supported lipid bilayer via vesicle fusion, and vesiclessuspended over membrane pores, but most of these are not able towithstand the high hydrostatic pressure that is required. Zhao's ownsolution to the problem is in effect to use a conventional membranepreparation as described above, modified by addition of aquaporin-loadedlipid vesicles (i.e. liposomes) to the aqueous solution of diamine. Theresult provides liposomes embedded in a polyamide layer. Although Zhaoreports the results obtained positively, it is clear from the dataprovided that although a small increase of water flux is obtained (FIG.4(a)) no enhancement of the ability of the membrane to reject solute isfound compared with conventional membranes (FIG. 5 ). It is believedthat this is because the aquaporin-loaded liposomes become completelysurrounded by polyamide, and thus the primary water flux through themembrane is via the polyamide (i.e. via the conventional path of thebase membrane), and only partially through the aquaporin channels. WO2013/043118, also from Zhao et al, describes the same technology andalso discloses that block copolymers can be used to form vesicles,either containing or not containing aquaporins, and embedded in apolyamide layer. Again, the results plainly show that water flux via thepolyamide layer and not exclusively via the aquaporin channels isobtained.

Xie et al, J. Mater. Chem A, 2013, 1, 7592, and WO 2013/180659, describea process comprising (i) incorporating aquaporin into self-assembledpolymer vesicles based on a polymer primarily (95%) having methacrylateend groups but also containing some (3%) carboxylic acid end groups;(ii) cross-linking the methacrylate end groups using UV light; (iii)depositing and covalently immobilizing the cross-linked vesicles on asupport in such a concentration that isolated vesicles are disposedseparately from each other on the surface of the support; and (iv)creating a thin polymer layer between the individual vesicles by theprocess known as “surface imprinting”. In this process, it is importantthat the size of the immobilized vesicles is such that they are largerthan the thickness of the imprinted polymer layer to prevent blockage ofthe aquaporin water channels. The process is said to exhibit highmechanical strength and stability during water filtration, but it isalso stated that the most critical issue is that the imprinted polymerlayer was not sufficiently dense to prevent all of the solute and watermolecules from permeating. Further, only very limited flow rates areobtainable by such a system.

Accordingly, there still remains a need for a process which leads to aphysically robust membrane incorporating transmembrane proteins,particularly a membrane which uses aquaporins acting effectively forwater filtration.

SUMMARY OF THE INVENTION

The invention provides a filtration membrane which comprises a poroussupport and, covalently bonded to a surface thereof, a layer comprisinga plurality of vesicles having transmembrane proteins incorporatedtherein, said vesicles being formed from an amphiphilic block copolymer;characterised in that within said layer, vesicles are covalently linkedtogether to form a coherent mass. The thickness of the layer will begreater than the average diameter of the vesicles. In absolute terms,the thickness of the layer is suitably at least 0.04 microns.

The invention further provides a process for the preparation of afiltration membrane according to the invention, which comprisesproviding an aqueous suspension of vesicles having transmembraneproteins incorporated therein, said vesicles being formed from anamphiphilic block copolymer having reactive end groups; depositing saidsuspension of vesicles on a surface of a porous support; and providingreaction conditions such that covalent bonds are formed betweendifferent vesicles and between vesicles and said surface.

Preferably, the filtration membrane is a water filtration membrane, andpreferably the transmembrane protein is an aquaporin. Throughout thisSpecification and claims, unless the context requires otherwise, anyreference to a filtration membrane should be understood to include aspecific reference to a water filtration membrane, and any reference toa transmembrane protein should be understood to include a specificreference to an aquaporin.

DETAILED DESCRIPTION OF THE INVENTION

In complete contrast to the process of Xie mentioned above, it is anessential feature of the present invention that the support carries alayer of vesicles in which multiple vesicles are close packed together.The packing in the layer may for example be hexagonal close packing. Thelayer of vesicles present on the support surface is thicker than theaverage diameter of the vesicles, i.e. it is of greater thickness thanwould be provided by a single layer of vesicles. It is preferred thatthe layer should have a thickness equivalent to at least 2, for exampleat least 10, preferably at least 50, more preferably at least 150, andmost preferably at least 200, times the average diameter of vesicles.Preferably the layer is not more than 500 times, for example not morethan 300 times, the average diameter of a vesicle. So, for example, thelayer may have a thickness of from 2 to 500, for example from 50 to 300,especially from 200 to 300 times the average diameter of the vesicles.In absolute terms, the thickness of the vesicle layer is preferably atleast 0.01, for example at least 0.04, for example at least 0.1, forexample at least 0.2, for example at least 2, preferably at least 10,more preferably at least 30, and most preferably at least 40, microns.There is no particularly preferred maximum thickness for the layer. Thelayer may for example have a thickness up to 100, for example up to 60,microns. So, for example, the layer may have a thickness of from 0.01 to100, for example from 0.04 to 100, for example from 0.2 to 100,preferably from 10 to 60, especially from 40 to 60, microns.

To increase robustness, the layer of vesicles in the finished membraneis preferably provided with a protective top coating layer, or a secondsupport layer on the opposite side from the support layer. This topcoating may for example provide added protection from mechanical damageduring a rolling process. It may for example comprise a hydrophilicpolymer, for example polyvinylalcohol.

The process of the invention may be carried out in a number of differentways. In a first preferred embodiment, there is provided a process forthe preparation of a membrane according to the invention, whichcomprises:

-   -   (a) providing an aqueous suspension of vesicles having        transmembrane proteins incorporated therein, said vesicles being        formed from amphiphilic block copolymers having reactive end        groups X;    -   (b) providing a multifunctional linking agent having at least        two reactive groups Y which are reactive with polymer end groups        X;    -   (c) depositing said suspension of vesicles and said        multifunctional linker on a support having a surface which is        reactive with either polymer end groups X or reactive groups Y;        and    -   (d) causing reaction of end groups X with groups Y, and either        end groups X or groups Y with the surface of the support.

In a second preferred embodiment, there is provided a process for thepreparation of a membrane according to the invention, which comprises:

-   -   (a) providing a first aqueous suspension of vesicles having        transmembrane proteins incorporated therein, said vesicles being        formed from amphiphilic block copolymers having reactive end        groups X;    -   (b) providing a second aqueous suspension of vesicles having        transmembrane proteins incorporated therein, said vesicles being        formed from amphiphilic block copolymers having reactive end        groups Y which are reactive with polymer end groups X;    -   (c) depositing said suspensions of vesicles on a support having        a surface which is reactive with either polymer end groups X or        Y; and    -   (d) causing reaction of end groups X with end groups Y, and        either end groups X or end groups Y with the surface of the        support.

The process of the invention results in a physically robust layer ofpolymer vesicles linked to each other, optionally via a linker, and alsolinked to the surface of the support.

It is not necessary that all the block copolymer molecules used in theinvention should have reactive end groups. The proportion of blockcopolymer molecules having reactive end groups is not critical, providedthat there are sufficient groups to react with reactive groups either ina second population of vesicles or in a multifunctional linker, to forma coherent mass. Generally, at least 10%, for example at least 20%, forexample at least 30%, for example at least 40%, for example up to 60%,or up to 100%, of the block copolymer molecules used to form thevesicles will have functional end groups X or Y. Similarly, it is notrequired that only one type of end group X or Y is present. It may forexample be desired to use blends of block copolymers, one containing onereactive end group X(1), for example an end group including an —NH₂group, and the second containing a different reactive end group X(2).

The end groups on any particular polymer molecule may be the same aseach other, or they may be different, but preferably they are the same.For example, one end group may be a reactive end group X, while theother end group may be a non-reactive group. The exact nature of thegroups will of course depend on the nature of the process and also onthe nature of the surface of the support.

Suitable reactive groups include amine groups (reactive with for examplecarboxylic acid, activated carboxylic acid and/or azide groups),carboxylic acid, activated carboxylic acid and/or azide groups (reactivewith for example amine groups Y), and “click chemistry” groups (forexample azide or alkyne groups, which are respectively reactive withalkyne and azide groups Y). The use of amine groups is particularlypreferred.

A wide variety of amine-based end groups is available, and these maycontain —NH₂ and/or —NH— groups. It has been found that when providingamphiphilic block copolymers with such end groups, the ability of theblock copolymer to self-assemble into vesicles is enhanced: this issurprising, as generally it is expected that the properties ofamphiphilic block copolymers which most influence vesicle formation are(i) the size and nature of the blocks; and (ii) the polydispersity ofthe polymer.

When using a multifunctional linking agent, the reactive groups presentin that agent may be the same as each other, or they may be different.They must be such as to react with complementary reactive groups presentin the vesicles and/or with the surface of the support. Suitable groupsare as mentioned above. When using a multifunctional reagent, thereagent may for example contain 3 or 4 reactive groups, but preferablyit contains two reactive groups, and any reference herein to amultifunctional reagent should be understood to include a specificreference to a difunctional reagent.

In a preferred embodiment of the invention, the vesicles containreactive groups which include an amine group; and a complementaryreactive group is provided which is an activated carboxylic acid groupor an azide, for example a phenylazide, group.

In one embodiment of the invention, the surface of the support may befunctionalised in one or more steps to introduce specific reactivegroups Z capable of reacting with complementary reactive groups X and/orY. Suitable groups include amine groups (reactive with for examplecarboxylic acid or activated carboxylic acid groups X and/or Y);carboxylic acid or activated carboxylic acid groups, (reactive with forexample amine groups X and/or Y); and “click chemistry” groups (forexample azide or alkyne groups reactive with alkyne or azide groups Xand/or Y). One example of a multi-step functionalization of a surface ishydrolysis of a polyacrylonitrile surface using acid, e.g. hydrochloricacid, to introduce surface carboxylic acid groups, which maysubsequently be activated using1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) andN-Hydroxysuccinimide (NHS) followed by conversion into alkyne groups,for example using propargylamine, or into azide groups, for exampleusing amino-triethyleneglycol-azide. However, in another embodiment ofthe invention, it may not be necessary to functionalise the surface ofthe support, because X and/or Y may be reactive with groups alreadypresent in the material forming the support. For example, Y may be anazide group: such groups are highly reactive once activated using UVlight, and are capable of reacting with C—H bonds present in manypolymers present in support materials. Specifically, azide, especiallyphenylazide, groups are capable of covalently bonding with polysulfones,which as discussed below, are a preferred support material for use inthe present invention.

Where reference is made to an activated carboxylic acid group, thisshould be understood to include any conventional activated carboxylicacid group, for example an activated ester such as anN-hydroxysuccinimide ester, or an acid halide. Such activationtechniques are well known in the art. In a preferred embodiment,activated carboxylic acid end groups are produced by the reaction of acarboxylic acid group with EDC and NHS. This is a well-known techniqueoften used in the world of protein conjugation and immobilization. Thereaction of a carboxyl group with EDC and NHS results in formation of anamine reactive NHS ester.

When using a multifunctional linker, its exact nature is not crucial,provided that it is capable of reacting efficiently to cause linking ofthe vesicles together by reaction of the X and Y groups.

Suitable multifunctional linkers include homobifunctional crosslinkers,that is, crosslinkers with the same functionalities at both ends.Examples which are capable of binding to amine groups include:

(i) NHS esters. Typical esters include:

-   -   disuccinimidyl glutarate:

-   -   bis(succinimidyl) polyethylene glycol:

for example bis(succinimidyl penta(ethylene glycol);

-   -   ethylene glycol bis(sulfosuccinimidylsuccinate):

-   -   3,3′-dithiobis(sulfosuccinimidylpropionate):

-   -   bis(sulfosuccinimidyl)suberate:

-   -   disuccinimidyl tartrate:

Reagents of this type react with primary amines in slightly alkalineconditions, for example at a pH of 7.2-8.5, for example 7.2-8.0, andyield stable amide bonds. Reaction temperatures are typically in therange of from 0 to 30, for example from 4 to 25° C. The reactionproduces N-hydoxysuccinimide which can be removed via dialysis ordesalting. The reaction may for example be carried out in PBS buffer atpH 7.2-8.0 for 0.5 to 4 hours at room temp or 4° C.

Sulfo NHS esters contain an —SO₃ group on the NHS ring. This has noeffect on the chemistry of the reaction, but such reagents tend to haveincreased water solubility.

(ii) Imidoesters. Typical imidoesters include the following (oftenobtained as dihydrochloride salts):

-   -   dimethyl adipimidate:

-   -   dimethyl 3,3′-dithiobispropionimidate:

-   -   dimethyl suberimidate:

-   -   dimethyl pimelimidate:

-   -   dimethyl adipimidate:

Imidoesters react with primary amines to form amidine bonds. To ensurespecificity for primary amines, the reaction is typically carried out inamine-free alkaline conditions (pH 9-11, for example pH10) with boratebuffer.

(iii) genipin, which has the formula:

(iv) epoxides, for example triglycidylamine:

(v) dialdehyde compounds, for example HOC.(CH₂)_(x).CHO, where x is 1 to6. Typical dialdehydes include glutaraldehyde, succindialdehyde,glyoxal, malondialdehyde, and phthalaldehyde.(vi) COOH-PEG-COGH. This reagent is water-soluble, and if desired may beactivated with EDC/NHS to provide reactivity with amines.

Suitable multifunctional linkers also include heterobifunctionalcrosslinkers, that is, crosslinkers with different functionalities atboth ends. Examples include:

1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (usually obtained in theform of the hydrochloride):

-   -   carbitol

-   -   epoxides, for example triglycidalamine;    -   COOH-PEG-NH₂;    -   sulfosuccinimidyl 6-(4′-azido-2′-nitrophenylamino)hexanoate;    -   poly(2-hydroxyethyl-co-2-methacryloxyethyl aspartamide);    -   N,N′-disuccinimidyl carbonate:

-   -   p-azidobenzoyl hydrazide:

The process of the invention may utilise “click chemistry”, which mayfor example utilise the reaction of an azide with an alkyne. Forexample, an alkyne group may be introduced as a group X or Y by reactionof a primary amine with an NHS ester. Many azide-PEG-azide linkers areavailable commercially.

Preferably a multifunctional linker includes a (CH₂)_(m) chain in whichm is from 2 to 20, preferably from 3 to 10, especially from 3 to 9. Anespecially preferred difunctional linker is the commercially availableproduct N-sulfosuccinimidyl-6-(4′-azido-2′-nitrophenylamino)hexanoate.This product has the formula:

The sulfosuccinimide group is a reactive group Y which is an activatedcarboxylic acid ester, capable of reacting spontaneously with aminegroups. The phenylazide group is a group Y which is inert underlight-free conditions, but becomes highly reactive when activated usingUV light, reacting readily with amine groups. In the absence of aminegroups, the activated group is also capable of reacting with groups of alower reactivity, even in some circumstances with a C—H bond;specifically, it is capable of reacting with the aromatic C—H groups ina polysulfone.

The conditions under which step (d) of the process of the inventiondescribed above, i.e. causing reaction of complementary reactive groupsX and Y, and reaction of either X or Y with the surface of the support,is carried out, will of course depend on the nature of the variousreactive groups. In some embodiments, the reactive groups will reactwith each other spontaneously once contacted together under suitableconditions. In other embodiments, photo-activatable groups may bepresent, in which case the reactants may be contacted together, andsubsequently photoirradiated to initiate reaction. In a preferredembodiment of the process of the invention, both mechanisms are combinedby using a multifunctional reagent having a first group Y which reactson contact with an end group X, and a second group Y which reacts withan end group X and with the surface of the support on irradiation withUV light.

Thus, the steps of one embodiment of the process of the invention may becarried out as follows:

-   -   (a) providing an aqueous solution of vesicles having        transmembrane proteins incorporated therein, said vesicles being        formed from amphiphilic block copolymers having reactive end        groups X;    -   (b) providing an multifunctional, preferably difunctional,        linking agent having at least two reactive groups Y which are        reactive with polymer end groups X, including a first reactive        group Y(1) being capable of reaction with polymer end groups X        under a first set of reaction conditions, and a second reactive        group Y(2) which is unreactive with polymer end groups X under        said first set of reaction conditions but which is reactive with        polymer end groups X under a second set of reaction conditions;    -   (b′) mixing said aqueous solution of vesicles with said        multifunctional linking agent under said first set of reaction        conditions so that reactive group Y(1) reacts with polymer end        groups X;    -   (c) depositing the resulting solution on a support which is        reactive with second reactive group Y(2), in an amount        sufficient to produce the desired layer of vesicles; and (d)        causing reaction of end groups X with said second reactive group        Y(2), and second reactive end groups Y(2) with the surface of        the support, by applying said second set of reaction conditions.

Any suitable reaction conditions which differentiate the two reactionsteps may be used. For example, the first set of reaction conditions mayinvolve groups X and Y(1) which react at a first temperature while thesecond set of reaction conditions may involve groups X and Y(2) whichreact at a second, higher, temperature. However, in a preferredembodiment, X and Y(1) are such that they react spontaneously oncontact, or with heating if necessary, while X and Y(2) are such thatthey react only when activated by photoirradiation. Accordingly, aparticularly preferred process comprises:

-   -   (a) providing an aqueous solution of vesicles having        transmembrane proteins incorporated therein, said vesicles being        formed from amphiphilic block copolymers having reactive end        groups X;    -   (b) providing a multifunctional, preferably difunctional,        linking agent having at least two reactive groups Y which are        reactive with polymer end groups X, including a first reactive        group Y(1) being capable of reaction with polymer end groups X        on contact, and a second reactive group Y(2) being capable of        reaction with polymer end groups X on photoirradiation;    -   (b′) mixing said aqueous solution of vesicles with said        multifunctional linking agent under conditions such that said        first reactive group Y(2) reacts with polymer end groups X;    -   (c) depositing the resulting solution on a support which is        reactive with second reactive group Y(2), in an amount        sufficient to produce the desired layer of vesicles; and    -   (d) applying photoirradiation to cause reaction of end groups X        with said second reactive group Y(2), and second reactive end        groups Y(2) with the surface of the support.    -   In a particularly preferred embodiment, the invention provides a        process which comprises:    -   (a) providing an aqueous solution of vesicles having        transmembrane proteins incorporated therein, said vesicles being        formed from amphiphilic block copolymers having terminal amine        groups;    -   (b) mixing said aqueous solution of vesicles with        N-sulfosuccinimidyl-6-(4′-azido-2′-nitrophenylamino)hexanoate        under conditions such that reaction between the succinimidyl        group of said hexanoate reacts with said polymer terminal amine        groups;    -   (c) depositing the resulting solution on a support; and    -   (d) irradiating said deposit with UV light to cause reaction of        the azide group of said hexanoate with polymer terminal amine        groups and with the surface of the support. Within this        embodiment, preferably the block copolymer is one of those        preferred polymers mentioned below, particularly        amine-terminated [(poly)2-methyl-2-oxazoline][(poly)dimethyl        siloxane][(poly)2-methyl-2-oxazoline].

In all the above embodiments, the amount of suspension deposited in step(c) is sufficient to provide the surface of the support with acontinuous layer of vesicles. Generally, after step (d) has been carriedout, this layer will be in the form of a coherent mass which has athickness greater than the average diameter of the vesicles; or, inabsolute terms, has a thickness of at least 0.04 microns.

A very wide range of reaction conditions may be used to effect theprocess of the invention. In one embodiment, when using amultifunctional linker, the quantity of multifunctional linker used willbe such that the total quantity of reactive groups Y present is inexcess of the total quantity of polymer end groups X present to ensureadequate crosslinking. Control of pH, temperature and other reactionconditions is conventional and within the normal practice of the skilledman.

The amphiphilic block copolymer is suitably a diblock copolymer ABhaving a hydrophilic and a hydrophobic block, or, preferably, a triblockcopolymer ABA having hydrophilic end blocks and a hydrophobic innerblock. The use of such copolymers in the formation of vesicles is wellknown, and a very wide range of hydrophilic polymers and hydrophobicpolymers may form the blocks A and B.

Hydrophobic polymers include for example polysiloxanes, for examplepolydimethylsiloxane or polydiphenylsiloxane, perfluoropolyether,polystyrene, polyoxypropylene, polyvinylacetate, polyoxybutylene,polyisoprene, polybutadiene, polyvinylchloride, polyalkylacrylates,polyalkylmethacrylates, polyacrylonitrile, polypropylene,polytetrahyrofuran, polymethacrylates, polyacrylates, polysulfones,polyvinylethers, and poly(propylene oxide), and copolymers thereof.

The hydrophobic segment preferably contains a predominant amount ofhydrophobic monomers. A hydrophobic monomer is a monomer that typicallygives a homopolymer that is insoluble in water and can absorb less than10% by weight of water.

Suitable hydrophobic monomers are dimethylsiloxanes, C₁-C₁₈ alkyl andC₃-C₁₈ cycloalkyl acrylates and methacrylates, C₃-C₁₈ alkylacrylamidesand -methacrylamides, acrylonitrile, methacrylonitrile, vinyl C₁-C₁₈alkanoates, C₂-C₁₈ alkenes, C₂-C₁₈ haloalkenes, styrene, (loweralkyl)styrene, C₄-C₁₂ alkyl vinyl ethers, C₂-C₁₀ perfluoro-alkylacrylates and methacrylates and correspondingly partially fluorinatedacrylates and methacrylates, C₃-C₁₂perfluoroalkylethylthiocarbonylaminoethyl acrylates and methacrylates,acryloxy- and methacryloxyalkylsiloxanes, N-vinylcarbazole, C₁-C₁₂ alkylesters of maleic acid, fumaric acid, itaconic acid, mesaconic acid,vinyl acetate, vinyl propionate, vinyl butyrate, vinyl valerate,chloroprene, vinyl chloride, vinylidene chloride, vinyltoluene, vinylethyl ether, perfluorohexyl ethylthiocarbonylaminoethyl methacrylate,isobornyl methacrylate, trifluoroethyl methacrylate,hexa-fluoroisopropyl methacrylate, hexafluorobutyl methacrylate,tristrimethylsilyloxysilylpropyl methacrylate, and3-methacryloxypropylpentamethyldisiloxane.

The hydrophobic polymer may include a single type of polymer or morethan one type of polymer, such as two or more of those mentioned above.

A preferred hydrophobic polymer is a polysiloxane, especially(poly)dimethylsiloxane.

The mean molecular weight (g/mol) of one segment B is in preferably inthe range from about 500 to about 50,000, preferably in the range fromabout 800 to about 15,000, more preferably in the range of about 1,000to 12,000, particularly preferably in the range from about 5,000 toabout 12,000.

In addition to the hydrophobic segment B, the amphiphilic copolymerincludes at least one, preferably two, segments A which include at leastone hydrophilic polymer, for example polyoxazoline, polyethylene glycol,polyethylene oxide, polyvinyl alcohol, polyvinylpyrrolidone,polyacrylamide, poly(meth)acrylic acid, polyethyleneoxide-co-polypropyleneoxide block copolymers, poly(vinylether),poly(N,N-dimethylacrylamide), polyacrylic acid, polyacyl alkylene imine,polyhydroxyalkylacrylates such as hydroxyethyl methacrylate,hydroxyethyl acrylate, hydroxypropyl acrylate, and polyols, andcopolymers thereof

The hydrophilic segment preferably contains a predominant amount ofhydrophilic monomers. A hydrophilic co-monomer is a monomer thattypically gives a homo-polymer that is soluble in water or can absorb atleast 10% by weight of water.

Suitable hydrophilic monomers include hydroxy 1-substituted lower alkylacrylates and methacrylates, acrylamide, methacrylamide, (lower alkyl)acrylamides and methacrylamides, N,N-dialkyl-acrylamides, ethoxylatedacrylates and methacrylates, polyethyleneglycol-mono methacrylates andpolyethyleneglycolmonomethylether methacrylates, hydroxyl-substituted(lower alkyl)acrylamides and methacrylamides, hydroxyl-substituted loweralkyl vinyl ethers, sodium vinylsulfonate, sodium styrenesulfonate,2-acrylamido-2-methylpropanesulfonic acid, N-vinylpyrrole,N-vinyl-2-pyrrolidone, 2-vinyloxazoline,2-vinyl-4,4′-dialkyloxazolin-5-one, 2- and 4-vinylpyridine, vinylicallyunsaturated carboxylic acids having a total of 3 to 5 carbon atoms,amino(lower alkyl)—(where the term amino also includes quaternaryammonium), mono(lower alkylamino)(lower alkyl) and di(loweralkylamino)(lower alkyl) acrylates and methacrylates, allyl alcohol.3-trimethylammonium 2-hydroxypropylmethacrylate chloride (Blemer, QA,for example from Nippon Oil), dimethylaminoethyl methacrylate (DMAEMA),dimethylaminoethylmethacrylamide, glycerol methacrylate, andN-(1,1-dimethyl-3-oxobutyl)acrylamide.

Specific examples of hydrophilic monomers from which such polymers canbe made are cyclic imino ethers, vinyl ethers, cyclic ethers includingepoxides, cyclic unsaturated ethers, N-substituted aziridines,β-lactones and β-lactams. Further suitable monomers include keteneacetals, vinyl acetals and phosphoranes. Suitable cyclic imino ethersinclude 2-oxazoline. If a 2-oxazoline having an alkenyl group in 2position is used as hydrophilic monomer, a polymerizable unsaturatedgroup is provided within segment A (in a side chain) of the amphiphilicsegmented copolymer to serve as the polymerizable unsaturated groupnecessary for the final polymerization to obtain a polymeric product oras an additional polymerizable unsaturated group which offers thepossibility of direct crosslinking in the preparation of the polymer.The most preferred cyclic imino ether is 2-C₁₋₃alkyloxazoline,especially 2-methyloxazoline. The most preferred vinyl ethers are methylvinyl ether, ethyl vinyl ether and methoxy ethyl vinyl ether.

A preferred hydrophilic polymer block is (poly)2-C₁₋₃alkyl-2-oxazoline,especially (poly)2-methyl-2-oxazoline.

The mean molecular weight (g/mol) of one segment A is suitably in therange from about 200 to about 50,000, preferably in the range from about800 to about 15,000, more preferably in the range of about 1,000 to12,000, particularly preferably in the range from about 5,000 to about12,000.

Synthesis of block copolymers by polymerisation is well known, and thelength of the one or more segments which are to be copolymerized on thestarting segment can be easily controlled by controlling the amount ofmonomer (hydrophilic or hydrophobic) which is added for thecopolymerization, and/or by the addition of suitable chain-terminatingcapping agents. In this way the size of the segments and their ratio caneasily be controlled.

As is well known in the art, the absolute and relative lengths of thehydrophilic and hydrophobic blocks are important in determining thesuitability of the copolymers for forming vesicles (so called polymerhydrophobic ratio). Further, the length of the blocks should be suchthat the thickness of the vesicle wall is broadly comparable with thelength of the transmembrane protein so that the protein can be readilyincorporated into the vesicle walls without the channel becomingblocked. For example the thickness of the vesicle wall may be in therange of from 1 nm to 50 nm. The length of the hydrophobic block B isparticularly important, and this should preferably be no greater than150 repeat units.

An especially preferred block copolymer for use in the present inventionis PAOXA-a-PDMS-b-PAOXA-a, especially PMOXA-a-PDMS-b-PMOXA-a, in whichPAOXA is (poly)2-C₁₋₃alkyl-2-oxazoline, PMOXA is(poly)2-methyl-2-oxazoline, and PDMS is (poly)dimethyl siloxane.Preferably each a independently is a number between 5 and 100,preferably between 10 and 100, and b is a number between 5 and 150,preferably between 20 and 150. Various PAOXA-PDMS-PAOXA polymers arecommercially available, and others can be readily synthesised by knownmethods.

Reactive end groups X may be present following initial synthesis of thecopolymer, or they may be introduced following the copolymer synthesis.For example, the copolymers may already contain suitable end groups whenusing a particular monomer, for example a dienepolymer such aspolybutadiene or polyisoprene, or if the monomer used for making ahydrophilic segment comprises an unsaturated side chain, for example2-allyl-oxazoline. Alternatively, the polymer may already containreactive end groups when the polymerisation has been stopped by use of asuitable capping agent. If not present following initial synthesis, itis possible to introduce reactive groups by suitable reactions at theend of the relevant block. For this purpose, the polymerization of thegrowing segment may be terminated after a suitable chain length isreached and the initiator group present at the chain end capped, forexample, either by using specific reagents such as hydroxy styrene,allyl alcohol, hydroxyethylmethacrylate, propargyl alcohol, allyl aminesand propargyl amine, or by using KOH/EtOH or primary amines leaving —OHor —NH— groups or unsaturated groups at the end of the growing segment.Hydroxyl groups may also be introduced into the copolymers by employingsuitable comonomers in the copolymerization, e.g.2-hydroxy-alkyloxazolines. The hydroxyl or —NH— groups may then bereacted, e.g. with an isocyanate carrying a polymerizable unsaturatedgroup. Preferred examples of such bifunctional compounds are vinylisocyanate, allyl isocyanate, acryloyl isocyanate, styrene isocyanate,vinyl benzyl isocyanate, and propargyl isocyanate. Other reactive groupscan be introduced by methods known to those skilled in the art.

In an especially preferred embodiment, the polymers used in the presentinvention have amine end groups. Most preferably the polymer isamine-terminated PAOXA-a-PDMS-b-PAOXA-a, for example one of thosePAOXA-PDMS-PAOXA polymers mentioned above, carrying amine end groups.

An amine end group may contain an —NH₂ group or an —NH— group, or both.In a particularly preferred embodiment of the invention, the amphiphilicblock copolymers are terminated by end groups X having the formula —NHRin which R represents an alkyl group which may be straight-chain orbranched having from 1 to 6 carbon atoms substituted by at least one,for example 1, 2 or 3, —NH₂ groups. Preferably such an end group X hasthe formula —NH—CH—(NH₂)₂ or, preferably, —NH—(CH₂)_(n)—NH₂, in which nis an integer from 2 to 6, preferably 2 to 4, especially 2. Such endgroups may be introduced by reacting a polymer having —OH end groupswith a suitable reactive amine NH₂R, for example a diamine, for exampleH₂N—(CH₂)_(n)—NH₂, especially H₂N—(CH₂)₂—NH₂, or triamine, for exampleN.([CH₂]_(n)NH₂)₃ or CH.([CH₂]_(n)NH₂)₃, for example CH(NH₂)₃ ortris(3-aminopropyl)amine. Branched oligomeric imines may also be used.

Amphiphilic block copolymers of the type PAOXA-a-PDMS-b-PAOXA-a, inwhich PAOXA is (poly)₂-C₁₋₃alkyl-2-oxazoline and PDMS is (poly)dimethylsiloxane, which contain end groups including both an —NH₂ group or an—NH— group, i.e. containing both a primary and a secondary amine group,especially —NH—CH—(NH₂)₂ or —NH—(CH₂)_(n)—NH₂, are novel and are claimedin our copending application reference no. 22883 WO. Vesicles formedfrom such polymers and having transmembrane proteins incorporatedtherein are also novel and claimed in our copending application.

Block copolymers can be prepared in the form of vesicles by methods wellknown in the art. Generally, these methods involve either solventdisplacement or solvent-free rehydration. In solvent displacementmethods, the block copolymer is dissolved in an organic solvent beforemixing with water. After mixing, and optionally removing the organicsolvent, spontaneous self-assembly of vesicles results. In solvent-freerehydration, dry block copolymer is brought into contact with an aqueousmedium whereupon hydration results in the spontaneous self-assembly ofvesicles. In a special case of solvent-free rehydration, the thin-filmrehydration process, block copolymer is dissolved in an organic solventwhich is then removed under conditions such that a thin film is formed.This film is then hydrated by contacting with water.

Vesicles having a desired size and low polydispersity can be obtained byknown methods, for example by extrusion of large uni- and multi-lamellarpolydisperse vesicles through one or more membranes of known pore size.Track etched polycarbonate membranes, for example Isopore (Trade Mark)membranes available from Millipore, are suitable for this purpose.Suitably, the vesicles used in the present invention have an averagediameter in the range of from 30 to 10,000, preferably 50 to 1000, morepreferably 100 to 400, especially from 150 to 250, nm.

The propensity of known PAOXA-a-PDMS-b-PAOXA-a polymers to formvesicles, rather than other self-assembly structures such as micelles,depends primarily on the absolute and relative sizes of the blocks.Thus, when the polymer is terminated with —OH groups, and when theblocks are relatively high molecular weight, for example as inPAOXA₁₄PDMS₅₅PAOXA₁₄ or higher, micelles tend to be formed, which meansthat lower molecular weight polymers need to be used if vesicles arerequired. Surprisingly, the presence of an end group including both an—NH₂ and an —NH—group makes a major difference, and the use ofPAMOXA-a-PDMS-b-PAOXA-a, for example PAOXA₁₄PDMS₅₅PAOXA₁₄ and inparticular PMOXA₁₄PDMS₅₅PMOXA₁₄ having such end groups, for example:

-   -   H₂N—(CH₂)_(n)—NH-PAOXA₁₄PDMS₅₅PAOXA₁₄-NH—(CH₂)_(n)—NH₂        particularly    -   H₂N—(CH₂)_(n)—NH-PMOXA₁₄PDMS₅₅PMOXA₁₄-NH—(CH₂)_(n)—NH₂        has proved particularly valuable for the preparation of        vesicles.

Overall, the use of functional group terminated polymers, particularlyamine-terminated polymers, together with a complementary multifunctionallinking agent gives major advantages compared with known processes forthe preparation of working filtration membranes.

However the vesicles are formed, the vesicle formation process can becarried out in the presence of transmembrane proteins, especiallyaquaporins, whereby the transmembrane protein becomes incorporated intothe wall of the vesicle. Generally, the process is carried out in thepresence of a detergent which assists in maintaining the integrity andbiological function of the protein. Thus, the above rehydration stepsmay be carried out using an aqueous solution of a transmembrane protein,preferably also including a detergent. The use of aquaporins ispreferred, and aquaporins are robust under a wide range of processconditions.

Aquaporins are biological cell transmembrane proteins whose function isto selectively transport water and no other molecules; the transportchannel of the protein is a two-way channel through which water can flowin either direction. They are expressed by many human cell types, andalso by bacterial and plant cells. Any of the different members of theaquaporin family of proteins can be used in the present invention.Suitable aquaporins include Aqp 4, Aqpl and, especially, Aqp Z.Aquaporins may exist in monomeric, dimeric, tetrameric and higheroligomeric forms, as well as mutated, conjugated and truncated versionsof the primary sequence. Provided that the biological function of theaquaporin, i.e. the selective transport of water, is maintained, any ofthese may be used in the present invention.

Any other transmembrane protein having desirable transport propertiesmay be used in the present invention. Variants of such transmembraneproteins, including naturally or non-naturally occurring variants andorthologs or paralogs of such proteins may be used. Such proteinsinclude for example:

-   -   Monotopic Membrane Proteins        -   Cyclooxygenases            -   Ram Prostaglandin H₂ synthase-1 (cyclooxygenase-1 or                COX-1): Ovis aries            -   Ram Prostaglandin H₂ synthase-1 (COX-1) R120Q/Native                Heterodimer: Ovis aries            -   Aspirin Acetylated COX-1            -   Cyclooxygenase-2: Mus Musculus        -   Squalene-Hopene Cyclases            -   Squalene-hopene cyclase: Alicyclobacillus acidocaldarius        -   Monoamine Oxidases            -   Monoamine Oxidase B: Human mitochondrial outer membrane            -   Monoamine Oxidase A: Rat mitochondrial outer membrane            -   Monoamine Oxidase A: Human mitochondrial outer membrane                -   G110A mutant        -   Hydrolases            -   Fatty acid amide hydrolase: Rattus norvegicus        -   Oxidoreductases (Monotopic)            -   Sulfide:quinone oxidoreductase in complex with                decylubiquinone: Aquifex aeolicus            -   Electron Transfer Flavoprotein-ubiquinone oxidoreductase                (ETF-QO): Sus scrofa        -   Peptidoglycan Glycosyltransferases            -   Peptidoglycan Glycosyltransferase: Staphylococccus                aureus            -   Peptidoglycan Glycosyltransferase penicillin-binding                protein 1a (PBP1a): Aquifex aeolicus            -   Peptidoglycan Glycosyltransferase penicillin-binding                protein 1b (PBP1b): Escherichia coli        -   Peptidases            -   Signal Peptidase (SPase): Escherichia coli            -   Signal Peptide Peptidase (SppA), native protein:                Eschericia coli        -   Dehydrogenases            -   Glycerol-3-phosphate dehydrogenase (GlpD, native):                Escherichia coli        -   Dihydroorotate Dehydrogenases (DHODH, class 2)            -   Dihydroorotate Dehydrogenase: Escherichia coli            -   Dihydroorotate Dehydrogenase: Rattus rattus            -   Dihydroorotate Dehydrogenase, apo form: Homo sapiens            -   Dihydroorotate Dehydrogenase: Plasmodium falciparum: 3d7        -   Polymerases            -   TagF teichoic acid polymerase: Staphylococcus                epidermidis        -   ADP-Ribosylation Factors            -   ADP-ribosylation factor (ARF1), myristoylated:                Saccharomyces cerevisiae            -   ADP-ribosylation factor (ARF1*GTP), myristoylated:                Saccharomyces cerevisiae        -   Isomerases            -   RPE65 visual cycle retinoid isomerase: Bos Taurus    -   Transmembrane Proteins: Beta-Barrel        -   Beta-Barrel Membrane Proteins: Multimeric            -   Porin: Rhodobacter capsulatus            -   Porin: Rhodopeudomonas blastica            -   OmpK36 osmoporin: Klebsiella pneumonia            -   Omp32 anion-selective porin: Comamonas acidovorans            -   Omp32 anion-selective porin: Delftia acidovorans            -   OmpF Matrix Porin: Escherichia coli            -   OmpC Osmoporin: Escherichia coli            -   OmpG *monomeric* porin: Escherichia coli            -   PhoE: Escherihia coli            -   LamB Maltoporin: Salmonella typhimurium            -   LamB Maltoporin: Escherichia coli            -   LamB Maltoporin: Escherichia coli            -   ScrY sucrose-specific porin: Salmonella typhimurium            -   MspA mycobacterial porin: Mycobacterium smegmatis            -   OprP phosphate-specific transporter: Pseudomonas                aeruginosa            -   OprD basic amino acid uptake channel: Pseudomonas                aeruginosa            -   OpdK hydrocarbon transporter: Pseudomonas aeruginosa            -   PorB outer membrane protein, native structure: Neisseria                meningitidis        -   Beta-Barrel Membrane Proteins: Monomeric/Dimeric            -   TolC outer membrane protein: Escherichia coli            -   TolC outer membrane protein, ligand blocked: Escherichia                coli            -   TolC outer membrane protein (Y362F, R367E): Escherichia                coli                -   C2 Form                -   P2:2:2 form            -   VceC outer membrane protein: Vibrio cholera            -   OprM drug discharge outer membrane protein: Pseudomonas                aeruginosa            -   CusC heavy metal discharge outer membrane protein:                Escherichia coli            -   CusBA heavy-metal efflux complex outer membrane protein:                Escherichia coli            -   BenF-like Porin (putative): Pseudomonasfluorescens            -   OprM drug discharge outer membrane protein: Pseudomonas                aeruginosa            -   apo BtuB cobalamin transporter: Escherichia coli            -   BtuB: Escherichia coli            -   apo BtuB by in meso crystallization: Escherichia coli            -   Colicin I receptor: Escherichia coli            -   OmpA: Escherichia coli, 2.5 Å            -   OmpA with four shortened loops: Escherichia coli                -   Called β-barrel platform (BBP)            -   OmpT outer membrane protease: Escherichia coli            -   Pla Plasminogen activator (native 1): Yersiniapestis            -   OmpW outer membrane protein: Escherichia coli                -   Orthorhomibic Form                -   Trigonal Form            -   OprG outer membrane protein: Pseudomonas aeruginosa            -   OmpX: Escherichia coli            -   TtoA Outer Membrane Protein (OMP): Thermus thermophilus                HB27            -   OmpLA (PldA) outer membrane phospholipase A monomer:                Escherichia coli            -   OmpLA (PldA) active-site mutant (N156A): Escherichia                coli            -   OpcA adhesin protein: Neisseria meningitidis            -   NspA surface protein: Neisseria meningitides            -   NalP autotransporter translocator domain: Neisseria                meningitides            -   NanC Porin, model for KdgM porin family: Escherichia                coli            -   Hia1022-1098 trimeric autotransporter: Haemophilus                influenza                -   Hia992-1098            -   EspP autotransporter, postcleavage state: Escherichia                coli            -   EstA Autotransporter, full length: Pseudomonas                aeruginosa            -   PagP outer membrane palimitoyl transferease: Escherichia                coli)            -   FadL long-chain fatty acid transporter: Escherichia coli            -   FadL long-chain fatty acid transporter A77E/S100R                mutant: Escherichia coli                -   ΔS3 kink                -   P34A mutant                -   N33A mutant                -   ΔNPA mutant                -   G212E mutant            -   FadL homologue long-chain fatty acid transporter:                Pseudomonas aeruginosa            -   FauA alcaligin outer membrane transporter: Bordetella                pertusssis TodX hydrocarbon transporter: Pseudomonas                putida            -   TbuX hydrocarbon transporter: Ralstoniapickettii            -   Tsx nucleoside transporter (apoprotein): Eschericia coli            -   FhuA, Ferrichrome-iron receptor: Escherichia coli            -   FepA, Ferric enterobactin receptor: Escherichia coli            -   FecA, siderophore transporter: Escherichia coli            -   HasR heme-uptake receptor: Serratia marcescens                -   Ile671Gly mutant            -   FptA, pyochelin outer membrane receptor: Pseudomonas                aeruginosa            -   FpvA, Pyoverdine receptor: Pseudomonas aeruginosa            -   FpvA, Pyoverdine receptor (apo form): Pseudomonas                aeruginosa            -   P pilus usher translocation domain, PapC130-640:                Escherichia coli        -   Beta-Barrel Membrane Proteins: Mitochondrial Outer Membrane            -   VDAC-1 voltage dependent anion channel: Human            -   VDAC-1 voltage dependent anion channel: Murine        -   Omp85-TpsB Outer Membrane Transporter Superfamily            -   FhaC Filamentous Hemagglutinin Transporter:                Bordetellapertussis            -   TeOmp85-N POTRA domains: Thermosynechococcus anaOmp85-N                Anabaena sp. PCC7120            -   BamA: Escherichia coli            -   BamE: Escherichia coli        -   Non-constitutive. Beta-sheet Pore-forming Toxins            -   Alpha-hemolysin: Staphylococcus aureus            -   LukF: Staphylococcus aureus            -   Perfringolysin O (PFO) protomer: Clostridium perfringens            -   Anthrax Protective Antigen (PA) and Lethal Factor (LF)                Prechannel Complex: Bacillus anthraciss            -   Lymphocyte preforin monomer: Mus musculus    -   Transmembrane Proteins: Alpha-Helical        -   Non-constitutive. Alpha-helical Pore-forming Toxins.            -   Cytolysin A (ClyA, aka HlyE): Escherichia coli            -   FraC eukaryotic pore-forming toxin from sea anemone:                Actiniafragacea        -   Outer Membrane Proteins            -   Wza translocon for capsular polysaccharides: Escherichia                coli            -   Porin B monomer: Corynebacterium glutamicum            -   Type IV outer membrane secretion complex: Escherichia                coli            -   Bacteriorhodopsin (BR): Halobacterium salinarium            -   Halorhodopsin (HR): Halobacterium salinarium            -   Halorhodopsin (HR): Natronomonas pharaonis            -   Sensory Rhodopsin I (SRI): Anabaena (Nostoc) sp. PCC7120            -   Sensory Rhodopsin II (SRII): Natronomonas pharaonis            -   Archaerhodopsin-1 (aR-1): Halorubrum sp. aus-1            -   Archaerhodopsin-2 (aR-2): Haloroubrum sp. aus-2            -   Xanthorhodopsin: Salinibacter ruber        -   G Protein-Coupled Receptors (GPCRs)            -   Rhodopsin: Bovine Rod Outer Segment (Bos Taurus)            -   Rhodopsin: Squid (Todarodes pacifzcus)            -   β1 adrenergic receptor (engineered): Meleagris gallopavo                (turkey)            -   β2 adrenergic receptor: Homo sapiens            -   Methylated β2 adrenergic receptor: Homo sapiens            -   A2A adenosine receptor: Homo sapiens            -   CXCR4 Chemokine Receptor: Homo sapiens            -   Dopamine D3 Receptor: Homo sapiens        -   Autonomously Folding “Membrane Proteins” (Sec-independent)            -   Mistic membrane-integrating protein: Bacillus subtilis        -   Glycoproteins            -   Glycophorin A transmembrane-domain dimer: Homo sapiens        -   SNARE Protein Family            -   Syntaxin 1A/SNAP-25/Synaptobrevin-2 Complex: ratus ratus        -   Integrin Adhesion Receptors            -   Human Integrin αIIbβ3 transmembrane-cytoplasmic                heterodimer: Homo sapiens        -   Histidine Kinase Receptors            -   ArcB (1-115) Aerobic Respiration Control sensor membrane                domain: Escherichia coli            -   QseC (1-185) Sensor protein membrane domain: Escherichia                coli            -   KdpD (397-502) Sensor protein membrane domain:                Escherichia coli        -   Immune Receptors            -   Transmembrane ζ-ζ dimer of the TCR-CD3 complex: Homo                sapiens            -   DAP12 dimeric: Homo sapiens        -   Channels: Potassium and Sodium Ion-Selective            -   KcsA Potassium channel, H+ gated: Streptomyces lividans            -   KcsA Potassium channel E71H-F103A inactivated-state                mutant (closed state): Streptomyces lividans            -   KcsA Potassium channel E711 modal-gating mutant:                Streptomyces lividans            -   KvAP Voltage-gated potassium Channel: Aeropyrum pernix            -   Kv1.2 Voltage-gated potassium Channel: Rattus norvegicus            -   Kv1.2/Kv2.1 Voltage-gated potassium channel chimera:                Rattus norvegicus                -   F233W Mutant            -   MthK Potassium channel, Ca++ gated: Methanothermobacter                thermautotrophicus            -   Human BK Channel Ca2+-activation apparatus: Homo sapiens            -   Kir3.1-Prokaryotic Kir Chimera: Mus musculus &                Burkholderia xenovornas            -   Kir2.2 Inward-Rectifier Potassium Channel: Gallus gallus            -   KirBac1.1 Inward-Rectifier Potassium channel:                Burkholderia pseudomallei            -   MlotiK1 cyclic nucleotide-regulated K+-channel:                Mesorhizobium loti            -   mGIRK1 G-Protein Gated Inward Rectifying Potassium                Channel: Mus musculus            -   NaK channel (Na+ complex): Bacillus cereus                -   D66/S70E Mutant                -   D66N Mutant                -   D66E Mutant            -   CNG-mimicking NaK channel mutant: Bacillus cereus            -   NaK channel; K+ selective mutant: Bacillus cereus        -   Channels: Other Ion Channels            -   GluA2 Glutamate receptor (AMPA-subtype): Rattus                norvegicus            -   M2 proton channel: Influenza A            -   M2 proton channel: Influenza B            -   ASIC1 Acid-Sensing Ion Channel: Gallus gallus            -   ATP-gated P2X4 ion channel (apo protein): Danio rerio                (zebra fish)            -   Nicotinic Acetylcholine Receptor Pore: Torpedo marmorata            -   Prokaryotic pentameric ligand-gated ion channel (pLGIC):                Erwinia chrysanthemi            -   Prokaryotic pentameric ligand-gated ion channel (GLIC):                Gloebacter violaceus                -   E221A mutant            -   Prokaryotic pentameric ligand-gated ion channel (GLIC),                wildtype-TBSb complex: Gloebacter violaceus                -   Wildtype-TEAs complex                -   E221D-TEAs complex                -   Wildtype-TMAs complex                -   Wildtype-bromo-lidocaine complex                -   Wildtype-Cd2+ complex                -   Wildtype-Zn2+ complex                -   Wildtype-Cs+ complex            -   MscL Mechanosensitive channel: Mycobacterium                tuberculosis            -   MscS voltage-modulated mechanosensitive channel:                Escherichia coli            -   CorA Mg2+ Transporter: Thermotoga maritime            -   MgtE Mg2+ Transporter: Thermus thermophilus            -   SLAC1 anion channel, TehA homolog (wild-type):                Haemophilus influenzae                -   F262A mutant                -   F262L mutant                -   F262V mutant                -   G15D mutant        -   Channels: Protein-Conducting            -   SecYEP protein-conducting channel:                Methanococcusjannaschii        -   Channels: Aquaporins and Glyceroporins            -   AQP0 aquaporin water channel: Bovine lens            -   AQP1 aquaporin water channel: Human red blood cell            -   AQP1 aquaporin water channel: Bovine red blood cell            -   AQP4 aquaporin water channel: rat glial cells                -   S180D Mutant            -   AQP4 aquaporin water channel: Human            -   AQP5 aquaporin water channel (HsAQP5): human            -   AqpM aquaporin water channel: Methanothermobacter                marburgensis            -   AqpZ aquaporin water channel: Escherichia coli            -   AqpZ aquaporin (C9S/C20S), T183C mutant: Escherichia                coli                -   L170C Mutant            -   AqpZ aquaporin mutant F43W: Escherichia coli                -   H17G/T183F Mutant                -   F43WH174G/T183F Mutant            -   SoPIP2; 1 plant aquaporin: Spinacia oleracea            -   GlpF glycerol facilitator channel: Escherichia coli            -   GlpF glycerol facilitator channel, W84F/F200T-mutant:                Escherichia coli            -   PfAQP aquaglyceroporin: Plasmodium falciparum:            -   Aqy1 yeast aquaporin (pH 3.5): Pischia pastoris        -   Channels: Formate Nitrate Transporter (FNT) Family            -   FocA, pentameric aquaporin-like formate transporter:                Escherichia coli            -   FocA formate transporter without formate: Vibrio                cholerae            -   FocA formate transporter: Salmonela typhimurium        -   Channels: Urea Transporters            -   Urea transporter: Desulfovibrio vulgaris            -   Connexin 26 (Cx26; GJB2) gap junction: Human        -   Channels: Amt/Rh proteins            -   AmtB ammonia channel (mutant): Escherichia coli            -   AmtB ammonia channel (wild-type): Escherichia coli                -   H168E Mutant                -   H168A Mutant                -   H168F Mutant                -   H318A Mutant                -   H318 Mutant                -   H318F Mutant                -   H168A/H₃₁₈A Mutant            -   Amt-1 ammonium channel: Archaeoglobusfulgidus            -   Rh protein, possible ammonia or CO2 channel:                Nitrosomonas europaea            -   Human Rh C glycoprotein ammonia transporter: Homo                sapiens        -   Intramembrane Proteases            -   GlpG rhomboid-family intramembrane protease: Eschericia                coli                -   W136A Mutant                -   S201T Active-Site Mutant            -   GlpG rhomboid-family intramembrane peptidase:                Haemophilus influenzae            -   Site-2 Protease (S2P). Intramembrane Metalloprotease:                Methanocaldococcus jannaschii            -   Signal Peptide Peptidase (SppA), native protein:                Escherichia coli        -   Membrane-Bound Metalloproteases            -   apo-FtsH ATP-dependent metalloprotease: Thermotoga                maritima        -   H+/Cl− Exchange Transporters            -   H+/Cl− Exchange Transporter: Salmonella typhimurium            -   H+/Cl− Exchange Transporter: Escherichia coli                -   E148A Mutant                -   E148Q Mutant                -   S107A/E148Q/445A Mutant            -   Monomeric H+/Cl− Exchange Transporter: Escherichia coli            -   +/Cl− Eukaryotic Exchange Transporter: Cyanidioschyzon                merolae            -   H+/Cl− Eukaryotic Exchange Transporter: Synechocystis                sp. pcc 6803        -   Bacterial Mercury Detoxification Proteins            -   MerF Hg(II) transporter: Morganella morganii        -   Multi-Drug Efflux Transporters            -   AcrB bacterial multi-drug efflux transporter:                Escherichia coli            -   AcrB bacterial multi-drug efflux transporter, apo                protein, N109A mutant: Escherichia coli            -   AcrB bacterial multi-drug efflux transporter, D407A                mutant: Escherichia coli            -   MexB bacterial multi-drug efflux transporter:                Pseudomonas aeruginosa            -   CusA metal-ion efflux pump: Escherichia coli            -   EmrE bacterial multi-drug efflux transporter:                Escherichia coli            -   NorM Multidrug and Toxin Compound Extrusion (MATE)                transporter (apo form): Vibrio cholerae        -   Membrane-Associated Proteins in Eicosanoid and Glutathione            Metabolism (MAPEG)            -   Microsomal Prostaglandin E Synthase 1: Human            -   5-Lipoxygenase-Activating Protein (FLAP) with Bound                MK-591 Inhibitor: Human            -   Leukotriene LTC4 Synthase: Human        -   Major Facilitator Superfamily (MFS) Transporters            -   LacY Lactose Permease Transporter (C₁₅₄G mutant):                Escherichia coli            -   LacY Lactose Permease (wild-type) with TDG: Escherichia                coli            -   FucP Fucose Transporter in outward-facing conformation:                Escherichia coli                -   N162A Mutant            -   GipT Glycerol-3-Phosphate Transporter: Escherichia coli            -   EmrD Multidrug Transporter: Escherichia coli            -   PepTSo Oligopeptide-proton symporter: Shewanella                oneidensis        -   Solute Sodium Symporter (SSS) Family            -   vSGLT Sodium Galactose Transporter:                Vibrioparahaemolyticus            -   K294A Mutant        -   Nucleobase-Cation-Symport-1 (NCS1) Family            -   Mhpl Benzyl-hydantoin transporter: Microbacterium                liquefaciens        -   Betaine/Choline/Carnitine Transporter (BCCT) Family            -   BetP glycine betaine transporter: Corynebacterium                glutamicum            -   CaiT carnitine transporter: Escherichia coli            -   CaiT carnitine transporter: Proteus mirabilis        -   Amino Acid/Polyamine/Organocation (APC) Superfamily            -   AdiC Arginine:Agmatine Antiporter: Escherichia coli                -   N22A, L123W Mutant                -   N101A Mutant            -   apo ApcT Na+-independent Amino Acid Transporter:                Methanocaldococcus jannaschii        -   Amino Acid Secondary Transporters            -   LeuTAa Leucine transporter: Aquifex aeolicus            -   Wild-type LeuT transporter: Aquifex aeolicus                -   E290S Mutant            -   Mutant LeuT transporter with Nitroxide Spin Label                (F177R1): Aquifex aeolicus                -   I204R1 Mutant            -   Glutamate Transporter Homologue (GltPh): Pyrococcus                horikoshii            -   Aspartate Transporter Li+-Bound State (GltPh):                Pyrococcus horikoshii        -   Cation Diffusion Facilitator (CDF) Family            -   YiiP Zinc Transporter: Escherichia coli        -   Antiporters            -   NhaA Na+/H+ antiporter: Escherichia coli            -   Mitochondrial ADP/ATP Carrier: Bovine heart mitochondria        -   Energy-Coupling Factor (ECF) Transporters            -   RibU, S Component of the Riboflavin Transporter:                Staphylococcus aureus        -   ATP Binding Cassette (ABC) Transporters            -   BtuCD Vitamin B12 Transporter: Escherichia coli            -   Sav1866 Multidrug Transporter: Staphylococcus aureus            -   Molybdate Transporter ModB2C₂: Archaeoglobusfulgidus            -   ModBC Molybdate ABC Transporter: Methanosarcina                acetivorans            -   HI1470/1 Putative Metal-Chelate-type ABC Transporter:                Haemophilus influenza            -   MsbA Lipid “flippase” with bound AMPPNP: Salmonella                typhimurium            -   P-Glycoprotein: Mus musculus (mouse)            -   MalFGK2-MBP Maltose uptake transporter complex:                Escherichia coli            -   MetNI Methionine uptake transporter complex: Escherichia                coli            -   FbpC ferric iron-uptake transporter nucleotide-binding                domain: Neisseria gonorrhoeae        -   Superfamily of K+ Transporters (SKT proteins)            -   TrkH potassium ion transporter: Vibrio parahaemolyticus            -   Calcium ATPase: Rabbit sarcoplasmic reticulum            -   Na,K-ATPase: Pig Kidney            -   Na,K-ATPase: Shark            -   Na,K-ATPase Regulatory Protein FXYD1: Human            -   Phospholamban homopentamer: Human sarcoplasmic reticulum            -   Plasma Membrane H+-ATPase: Arabidopsis thaliana        -   V-type ATPase            -   Rotor of V-type Na+-ATPase: Enterococcus hirae            -   V1-ATPase Complex: Thermus thermophiles            -   A3B3 complex of V1-ATPase: Thermus thermophilus        -   F-type ATPase            -   F1-ATPase from bovine heart mitochondria: Bos Taurus            -   ATP synthase (F1c10): S. cerevisiae            -   F1 ATPase: S. cerevisiae            -   Rotor (c11) of Na+-dependent F-ATP Synthase: Ilyobacter                tartaricus            -   Rotor (c14) of H+-dependent F-ATP Synthase of spinach                chloroplasts: Spinacia oleracea            -   Rotor (c15) of H+-dependent F-ATP Synthase of an                alkaliphilic cyanobacterium: Spirulina platensis            -   Rotor (c13) of H+-dependent F-ATP Synthase: Bacillus                pseudofirmus            -   Peripheral stalk of H+-dependent F-ATP Synthase: Thermus                thermophilus        -   Phosphotransferases            -   Diacylglycerol kinase (DAGK): Escherichia coli        -   Hydrolases            -   Estrone Sulfatase: Human placenta        -   Oxygenases            -   Particulate methane monooxgenase (pMMO): Methylococcus                capsulatus            -   Particulate methane monooxgenase (pMMO): Methylosinus                trichosporium OB3b        -   Oxidoreductases            -   Sulfide:quinone oxidoreductase: Aquifex aeolicus            -   Electron Transfer Flavoprotein-ubiquinone oxidoreductase                (ETF-QO): Sus scrofa            -   Glycerol-3-phosphate dehydrogenase (GlpD, native):                Escherichia coli            -   NarGHI Nitrate Reductase A: Escherichia coli                -   K86A Mutant                -   H₆₆Y Mutant            -   NrfH Cytochrome C Quinol Dehydrogenase: Desulfovibrio                vulgaris            -   DsbB-DsbA Periplasmic Oxidase Complex: E. coli            -   DsbB-Fab complex: Eschericia coli            -   wtDsbB-DsbA (Cys133A)-Q8 Complex: E. coli            -   Vitamin K epoxide reductase: Synechococcus sp.        -   Mo/W bis-MGD Oxidoreductases            -   Polysulfide Reductase PsrABC (native): Thermus                thermophiles        -   Electron Transport Chain Complexes: Complex I            -   Complex I membrane domain: Escherichia coli            -   Complex I complete: Thermus thermophiles        -   Electron Transport Chain Complexes: Complex II            -   Native Fumarate Reductase Complex: Escherichia coli            -   Fumarate Reductase Complex: Wolinella succinogenes            -   Formate dehydrogenase-N: Escherichia coli            -   Succinate dehydrogenase (Complex II): Escherichia coli            -   Succinate:ubiquinone oxidoreductase (SQR, Complex II):                porcine heart mitochondria            -   Succinate:ubiquinone oxidoreductase (SQR, Complex II):                chicken heart mitochondria        -   Electron Transport Chain Complexes: Complex III (Cytochrome            bcl)            -   Cytochrome bcl: Bos Taurus            -   Cytochrome bel: Gallus gallus            -   Cytochrome bel: Sarcomyces cerevisiae            -   Cytochrome bcl: Rhodobacter Sphaeroides        -   Electron Transport Chain Complexes: Cytochrome b6f of            Oxygenic Photosynthesis            -   Cytochrome b6f complex: Mastigocladus laminosus            -   Cytochrome b6f complex: Chlamydomonas reinhardtii            -   Cytochrome b6f complex: Nostoc sp. PCC 7120        -   Electron Transport Chain Complexes: Complex IV (Cytochrome C            Oxidase)            -   Cytochrome C Oxidase, aa3: Bos taurus (bovine) heart                mitochondria            -   Cytochrome C Oxidase, aa3: Paracoccus denitrificans                -   N131D Variant            -   Cytochrome Oxidase, cbb3: Pseudomonas stutzeri            -   Cytochrome ba3: Thermus thermophilus            -   Cytochrome C Oxidase wild-type: Rhodobacter sphaeroides            -   Ubiquinol Oxidase, cytochrome bo3: E. coli        -   Nitric Oxide Reductases            -   Nitric Oxide Reductase: Pseudomonas aeruginosa        -   Photosystems            -   Photosystem I: Thermosynechococcus elongates            -   Photosystem I (plant): Psium sativum            -   Photosystem II: Thermosynechococcus elongates            -   Photosystem II: Thermocynechococcus vulcanus        -   Light-Harvesting Complexes            -   Light-Harvesting Complex: Rhodopseudomonas acidophila            -   Light-Harvesting Complex: Rhodospirillum molischianum            -   Light-Harvesting Complex LHC-II, Spinach Photosystem II:                Spinacia oleracia            -   Light-Harvesting Complex CP29, Spinach Photosystem II:                Spinacia oleracia            -   Light-Harvesting Complex LHC-II, Pea Photosystem II:                Pisum sativum        -   Photosynthetic Reaction Centers            -   Photosynthetic Reaction Center: Blastochloris viridis            -   Photosynthetic Reaction Center: Rhodobacter sphaeroides            -   Photosynthetic Reaction Center: Thermochromatium tepidum

The support may be made of any suitable microporous material. It may forexample be based upon a conventional membrane support, as used inreverse osmosis or ultrafiltration membranes. Such supports may forexample be made from a polyolefin, cellulose, regenerated cellulose,cellulose acetate, polyacrylonitrile, polyethersulfone, or polysulfone.In a preferred embodiment of the invention, the support is made from apolysulfone.

Chemical functionality of the support membrane may be delivered in theform of additives, which may be either low molecular weight orpolymeric, to the casting dope, or functionalization of the supportsurface, for example by chemical treatments, graft polymerisation orplasma polymerization. By these means, the following chemicaltransformations of the support may for example be accomplished:conversion of amine groups into carboxylic acid groups, or vice versa;conversion of aldehydes into amines; and conversion of hydroxyl groupsinto carboxylic acid groups. All such reactions are well known in theart.

Porous ultrafiltration membranes may for example be prepared by aircasting, where the dissolved polymer solution passes under a series ofair flow ducts that control the evaporation of the solvents in a veryslow manner; solvent or emersion casting, where the dissolved polymer isspread onto a moving belt and run through a bath of liquid, and theliquid in the bath exchanges with the solvent in the lacquer and causesthe formation of the pores; thermal casting, where heat is used to drivethe solubility of the polymer in a given solvent system. The lacquer isthen cast out onto a moving belt that is being cooled. Quenching theheat in the lacquer causes precipitation to start and the pores to form.Materials typically used in the process include but are not limited tocellulose regenerated, cellulose nitrate, cellulose acetate, polyamide,polysulfone, poly(ether sulfone), polycarbonate, poly(ether imide),poly(2,6-dimethyl-1,4-phenylene oxide), polyimide, poly(vinylidenefluoride), polytetrafluoroethylene, polypropylene, polyacrylonitrile,poly(methyl methacrylate, polyvinyl alcohol, and polydimethylsiloxane.The morphology of the cast is regulated by the configuration of thefinal module. It may for example comprise a flat-sheet for spiral woundelements; hollow-fibre for hollow-fibre elements; or it may be tubular.

Preparation of a membrane having a layer comprising a coherent mass ofvesicles, said layer having a defined thickness, may be achieved bycontrol of the concentration of vesicles present in the solution ofvesicles applied to the support and/or by the volume of solutiondeposited on the support.

Xie et al, J. Mater. Chem A, 2013, 1, 7592, discloses processesinvolving crosslinking during the preparation of the polymer vesicles,but this crosslinking, which did not change the structure or dimensionof the polymer vesicles (col. 2 p. 7596 top paragraph) is alwaysinternal crosslinking between the crosslinkable end groups correspondingto the groups X of the present invention. Similarly, the crosslinkingdisclosed in WO 01/32146 is always internal crosslinking. It is ofcourse possible, depending on the nature of the various groups present,for internal cross-linking to occur in the vesicles of the presentinvention, but it is an essential feature of the present invention thatexternal crosslinking, preferably via a multifunctional linker, alsotakes place. The advantage of the present invention over the methodsdisclosed by Xie et al, and by Zhao et al, J. Membrane Sci. 2012,422-428 and WO 2013/043118, is that any possible pathway through themembrane other than through the transmembrane proteins embedded in thewalls of the polymer vesicles, is minimised, while providing a largenumber of possible transmembrane proteins per unit surface area of thesupport membrane, thus maximising flux through the membrane. The processis technically simple, and the resulting membranes are physicallyrobust.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the NMR spectrum of the polymer prepared in step 1 ofExample 1.

FIG. 2 shows the results of the molecular weight cut-off experiments ofstep 4 of Example 2.

FIG. 3 shows the results of the flow testing experiments of step 5 ofExample 1.

FIGS. 4 and 5 are scanning electron microscopy images of the membranesprepared in Example 1.

FIG. 6 shows the results of the dynamic light scattering measurements ofExample 2.

FIGS. 7A and 7B show LSM imaging micrographs of vesicles prepared inExample 4.

FIG. 8 shows the effect of incorporating Aquaporin Z protein intovesicles as described in Example 4.

FIG. 9 is a micrograph of the membrane of Example 4.

FIG. 10 shows the effect of internally cross-linking the polybutadienein the membrane of Example 4.

The following Examples illustrate the invention.

Example 1

Materials:

-   -   2-methyl-2-oxazoline, Sigma    -   Triethylamine, Sigma    -   Hexane, anhydrous, Sigma    -   Ethylene diamine, Sigma    -   Trifluoromethanesulfonic acid, Sigma    -   Ethyl acetate, Sigma    -   Aquaporin-Z stock solution 1 mg/ml in 1% octyl glucoside and 100        mM NaMPOS buffer at pH 7.5    -   100 mM NaMPOS buffer at pH 7.5    -   Chloroform (Puriss)    -   Octyl glucoside (Anatrace)    -   Amine functional polymer vesicles 10 mg/mL in Na.MOPS    -   PoPR (Polymer to Protein ratio, mass)    -   N-sulfosuccinimidyl-6-(4′-aizido-2′-nitrophenylamino)hexanoate,        sulfo-SANPAH (Pierce; Product No. 22589)    -   Dextrans (American Polymer Standards Corporation)    -   365 nm UV lamp (Entela UVP)    -   47 mm Membrane stamp    -   25 mm Membrane stamp    -   Polysulfone membrane; pore size 150 nm (cut-off over 1000 kDa)        1) Polymer preparation—Primary/Secondary-Amine terminated        poly-2-methyloxazoline-poly-di-methyl-siloxane-poly-2-methyloxazoline        (PMOXA-PDMS-PMOXA)

Step a). α,ω-Hydroxy-Butyl-Poly-Di-Methyl-Siloxane (PDMS) Synthesis:

Targeting the molecular weight of 4000 g/mol, 93.03 μg (0.34 mols) ofoctamethylcyclotetrasiloxane and 6.97 g (0.0025 mols)1,3-bis(hydroxybutyl)-tetramethyldisiloxane were charged into a 3-neckedround bottom Pyrex reactor with an argon inlet, thermometer andcondenser. Trifluoroacetic acid 6.55 g (0.05755 mols) was added. Thereaction mixture was heated at 60° C. for 48 hours. After this time theexcess trifluoroacetic acid was extracted with distilled water until theaqueous extract was neutral. Then the reaction mixture was stripped offunder high vacuum to remove the cyclic side products. Ester groups werefurther converted to alcohols by a weak base catalyzed hydrolysis in THFand an equal volume of 5% aqueous sodium carbonate solution at 40-45°C., for 48 hours. Organic and aqueous phases were separated out. The83.72 grams of product were recovered by the evaporation of THF. Theproduct was evaluated for molecular weight by proton NMR and molecularweight distribution by GPC in chloroform.

Step b). Primary/Secondary-Amine Terminated PMOXA-PDMS-PMOXA Synthesis

Hydroxyl-terminated PDMS synthesized as in step a above was used in thesynthesis of poly PMOXA-PDMS-PMOXA amphiphilic block copolymer.

In a three-neck round bottom flask 50 grams (0.012 mols) of PDMS werekept under high vacuum for 24 h. In the next step, a reaction flask wasfilled with dry argon, and the polymer was dissolved in dry hexane (200ml) and added to the three-neck flask via septum. Cooled (0-5 deg C.)PDMS was than activated by drop-wise addition of 6.62 g (0.02346 mols)of trifluoromethanesulfonic anhydride in presence of 2.45 g (0.024 mols)of triethyl amine and allowed to post-react for 3 hours. The activatedPDMS was further filtered under argon and hexane was removed underreduced pressure. 250 ml of dry ethyl acetate was added to re-dissolvethe activated polymer, and ring-opening polymerization of2-methyloxazoline was started upon addition of 23.5 g (0.27 mols) dried2-methyl oxazoline at 40 deg C. After 12 hours reaction under argon, a3-fold excess, 4.14 g (0.069 mols) of butyl-di-amine was added asterminating agent. Product was recovered under high vacuum and evaluatedfor molecular weight by proton NMR (shown in FIG. 1 ) and molecularweight distribution by GPC in chloroform. The product was 100% solublein ethanol and 99.5% insoluble in hexane. The remaining 0.5% was foundto be unreacted PDMS as shown by proton NMR.

2). Polymer Vesicles/Proteo-Vesicles Preparation:

50 mg of ABA block-co-polymer was dissolved in 2 ml of chloroform in around bottom flask (Pyrex 100 ml). Chloroform was then removed underhigh vacuum to form a thin film of polymer. This film was hydrated witheither 5 ml of buffer (control) or 5 ml of aqueous stock solution ofAquaporin-Z and stirred overnight. In these samples the amount of addedprotein was varied from 1:1 to 1:1200 polymer to protein ratio.Detergent was subsequently removed by dialysis in 30 kDa dialysismembranes in NaMOPS buffer. The resulting product was then extrudedthrough track-etched membranes to uniform 200 nm size.

3). Coating

In this step, the concentration of deposited vesicles was kept constantand monitored by matching the count rate (250 kcps) in Dynamic LightScattering (Malvern Zetasizer Nano) with static attenuator.

Sulfo-SANPAH (SS) solution (10 mM in 100 mM NaMOPS pH 7.5) was allowedto react with vesicles prepared as in step (1) in the absence of light(250 μL of vesicle solution combined with 50 μL SS for 15-minutes). Aseries of 47 mm polysulfone membranes (Nano H₂O Inc, 150 nm) were cut bypunch press and placed into Teflon membrane holders and rinsed withdeionized water. Excess water was removed by compressed air and 300 μL(each) of SS-activated vesicles/proteo-vesicles solutions were placedonto polysulfone support membranes. The membrane holders were thenplaced under UV light approximately 5 cm from the source and coveredwith foil for protection for 30 minutes. Excess reactants were thenremoved from the membrane surface using a 1 ml pipette without touchingthe membrane surface. The above steps were repeated three times,following which the membranes were removed from the holders and 25 mmdiameter membrane samples were cut from the coated area using a punchpress. These were then rinsed in excess 100 mM NaMOPS ph7.5 on a shaketable for at least one hour before testing.

4) Molecular Cut-Off Experiments

The 25 mm samples of step (2) tested for their ability to retain highmolecular weight materials, by measuring their molecular weight cut-off,i.e. the point at which at least 90% of molecules of a given molecularweight are retained by the membrane.

Phosphate buffer (0.03M Na₂HPO₄+0.03M KH₂PO₄) was pre-filtered using a0.2 um membrane and the pH was adjusted to 7.2 prior to use forpreparation of solutions. Dextran (DXT) standards were dissolved inphosphate buffer (DXT 165 kDa, 325 kDa, 548 kDa, 1300 kDa, and 5000 kDa,DXT 0.505 kDa, 4 kDa, 6 kDa, 11 kDa, 20 kDa, and 28 kDa). All of thedextran solutions were diluted to 0.5 mg/ml with phosphate buffer andpre-filtrated using a 0.2 um PES membrane prior to use. All filtrationexperiments were conducted in a 10 ml Amicon stirred ultrafiltrationcell (Model 8010, Millipore Corp.)

All samples were evaluated according to the protocol described below:

-   -   Filtered 10 ml volume of deionised water at 20 psi to wet the        pore structure and the whole system.    -   Connected the feed line with dextran solution feed to a digital        peristaltic pump (Thermal Fisher Science Inc.), re-pressurized        the cell to 20 psi, set the filtrate flux to 5 μm/s.    -   Obtained 800 μL samples of the filtrate solution after        filtration of 2,000 μL of water for equilibration and washing        out the dead volume downstream of the membrane.    -   Obtained 1 ml permeate samples directly from the cell after        filtration.    -   Cleaned and rinsed the whole system with deionised water.    -   The stirring speed was kept at 600 rpm and all experiments were        performed at room temperature (22±3° C.)

Permeate was further evaluated using high-pressure liquid chromatography(HPLC columns PL1149-6840, MW 10,000 to 200,000, PL1120-6830, MW 100 to30,000, PL1149-6860, MW 200,000 to >10,000,000). Comparison of the feedto the permeate chromatograms allowed for calculation of retentioncoefficients and membrane molecular cut-off.

The results are shown in FIG. 2 , which shows that all of the membranesaccording to the invention retained all of the higher molecular weightmolecules, while the control membrane demonstrated significantly poorerperformance, with a molecular weight cut-off in excess of 3,000 kDa.

5). Flow Testing

The 25 mm membranes of Step (2) were tested for their ability totransmit pure water using a stirred test cell (Amicon 10 ml, (Model8010, Millipore Corp.) in which the feed was pure water. The system wasclosed and set to stir for at least 5 min before testing. Subsequentlythe pressure was gradually increased from 1 to 5 bar and data pointsrepresenting the volume of pure water passing through the surface of themembrane in 1 minute were collected at 1 bar intervals (with permeatecollected separately at each pressure). The experiment also included thebest commercially available water filtration membrane currently on themarket, Biomax 30 kDa from Millipore, for comparison.

The results are shown in FIG. 3 , in which LMH/bar is litre/m²/hour/barof pure water, i.e. is a pressure-corrected flow rate, and PoPrrepresents polymer:protein ratio (note that the higher the PoPr, thelower the content of aquaporin protein).

The control membrane prepared in step 2 with a coating of vesicles butno aquaporin protein, had the lowest flow rate of all the membranestested. All the membranes according to the invention performedsignificantly better, with a higher content of aquaporin leading tohigher fluxes, and the membrane with the highest content of aquaporinsignificantly outperforming the commercially available membrane.

FIGS. 4 and 5 show SEMs of the membranes according to the invention. InFIG. 4 (magnification 1000) the lower layer having a sponge-likeappearance is the polysulfone support, having a macrovoid due to thecasting process. The upper layer is the continuous coating comprising acoherent mass of aquaporin-containing vesicles. In FIG. 5 (magnification20,000), the lower portion of the SEM having a textured appearance isthe polysulfone support, while the thin uppermost layer is thecontinuous coating comprising a coherent mass of aquaporin-containingvesicles. The bright line at the boundary between these two layers is aboundary layer where the vesicle layer is covalently bound to thepolysulfone.

Examples 2 and 3

Model experiments were carried out to confirm the suitability of variouspolymer end-groups for the preparation of vesicles and the covalentlinking of vesicles to each other. The alternative polymers wereprepared as follows.

(a) Carboxylic-terminatedpoly-2-methyloxazoline-poly-di-methyl-siloxane-poly-2-methyloxazoline(PMOXA-PDMS-PMOXA)

Hydroxyl-terminated polymer Mn=4262 g/mol (PDMS) synthesized as in step(a) of Example 1 was used in the synthesis of poly PMOXA-PDMS-PMOXAamphiphilic block copolymer.

In a three-neck round bottom flask 50 grams (0.01173 mols) of PDMS waskept under high vacuum for 24 h. In the next step reaction the flask wasfilled with dry argon and polymer was dissolved in dry hexane (200 ml)added to the three-neck flask via septum. Cooled (0-5 deg C.) PDMS wasthan activated by drop-wise addition of 6.62 g (0.02346 mols) oftrifluoromethanesulfonic anhydride in presence of 2.45 g (0.024 mols) oftriethylamine and allowed to post-react for 3 hours. The activated PDMSwas then filtered under argon and hexane was removed under reducedpressure. 250 ml of dry ethyl acetate was added to re-dissolve theactivated polymer and ring-opening polymerization of 2-methyloxazolinewas started upon addition of 23.5 g (0.27 mols) dried 2-methyl oxazolineat 40 deg C. After 12 h reaction under argon, deprotonated malonic acidwas added in 1.3× excess as terminating agent 3.12 g (0.030 mols) in thepresence of trietylamine 3.05 g (0.030 mols). Product was recoveredunder high vacuum and evaluated for molecular weight by proton NMR andmolecular weight distribution by GPC in chloroform.

(b) Hydroxy terminatedpoly-2-methyloxazoline-poly-di-methyl-siloxane-poly-2-methyloxazoline(PMOXA-PDMS-PMOXA)

Hydroxyl-terminated silicon Mn=4262 g/mol (PDMS) synthesized asdescribed in step (a) of Example 1 above was used in the synthesis ofpoly PMOXA-PDMS-PMOXA amphiphilic block copolymer.

In a three-neck round bottom flask 50 grams (0.01173 mols) of PDMS waskept under high vacuum for 24 h. In the next step reaction flask wasfilled with dry argon and polymer was dissolved in dry hexane (200 ml)added to the three-neck flask via septum. Cooled (0-5 deg C.) PDMS wasthen activated by drop-wise addition of 6.62 g (0.02346 mols) oftrifluoromethanesulfonic anhydride in the presence of 2.45 g (0.024mols) of triethylamine and allowed to post-react for 3 hours. Theactivated PDMS was then filtered under argon and hexane was removedunder reduced pressure. 250 ml of dry ethyl acetate was added tore-dissolve activated polymer and ring-opening polymerization of2-methyloxazoline was started upon addition of 23.5 g (0.27 mols) dried2-methyl oxazoline at 40 deg C. After 12 h reaction under argon,potassium hydroxide was added in 1.3× excess as terminating agent (1.68g (0.030 mols) in 50 ml of methanol). Product was recovered under highvacuum and evaluated for molecular weight by proton NMR and molecularweight distribution by GPC in chloroform.

Example 2

250 μL of vesicles made from amine-terminated polymer as prepared inExample 1 were placed in a 64 mL clear glass vial, and protected fromlight by wrapping the vials in aluminum foil. The varying amounts (0, 1,5, 10, 25 and 50 μl) of the difunctional linker sulfo-SANPAH, (10 mMSulfo-SANPAH in 100 mM Na.MOPS pH 7.5) was added and mixed by gentleshaking. Reaction was allowed to take place for 15 minutes, followingwhich 100 μL of solution was placed into a cuvette for dynamic lightscattering (DLS) measurement, DLS being a technique for the measurementof the size of particles in solution. The sample was placed about 5 cmbelow the UV lamp, the lid and foil were removed, the lamp was switchedon, and the whole was covered with a foil tent. In all cases theattenuator was fixed at 6. After 15 minutes under UV,

Prior to reaction with sulfo-SANPAH, DLS showed the diameter of thevesicles to be 200 nm. After UV irradiation to cause reaction withsulfo-SANPAH, large aggregates were formed which could be seen with thenaked eye. The DLS results are shown in FIG. 6 . These aggregates werestable under sonication, indicating the presence of covalent bonding.

As a comparison, a similar experiment was carried out usinghydroxyl-terminated polymer, which is not expected to be reactive withsulfo-SANPAH. As expected, no crosslinking occurred, and therefore noincrease in diameter measured by DLS occurred.

Example 3

Experiments were carried out using vesicles made from polymers havingactivated carboxylic acid groups as end groups.

Materials

-   -   EDC, Pierce (Product No. 22980)    -   NHS, Pierce (Product No. 24500)    -   Malvern ZetasizerNANO DLS    -   Sonication Bath    -   pH Meter with micro probe    -   Carboxyl terminated polymer vesicles prepared as described above    -   Amine terminated polymer vesicles prepared as described above

EXPERIMENTAL

Vesicles were prepared according to above described thin-film hydrationprotocol using deionised water. The average diameter of the resultingpolymer vesicles was shown to be around 200 nm using DLS.

Carboxylic vesicles activated with EDC and NHS were prepared by additionof 950 μg of EDC and 570 μg of NHS to 1 ml of carboxylic vesicles. Thesolution was then adjusted to pH 5 using HCl and allowed to react for 30minutes at room temperature resulting in EDC-NHS activated vesicles.

Solutions of (control) carboxylic vesicles (1 ml) and EDC-NHS activatedvesicles (1 ml) were allowed to react with equal amount ofamine-functional vesicles (1 ml). Subsequently the pH of all solutionswas adjusted about 7.5 with a dilute solution of NaOH in deionised waterand allowed to react for at least 90 minutes. 100 μL of the resultingsamples were tested by DLS using a static attenuator setting of 5. Aftertesting, the cuvettes were sonicated for 1 minute and then retested.

It was found that reaction of equal amounts of amine and carboxylicvesicles resulted in the formation of large aggregates (around 2000 nmby DLS). However, when sonicated, these aggregates dispersed, showingthat the bonding was ionic rather than covalent. In contrast, reactionof equal amounts of amine and EDC-NHS activated carboxylic vesiclesresulted in formation of large aggregates (about 3600 by DLS) which werenot dispersed when sonicated, indicating that the forces holdingaggregates together were covalent.

Example 4

A series of experiments using the diblock copolymer polybutadiene-PMOXAwas carried out.

Step (a): PB Synthesis

Polybutadiene was synthesized following the protocol of Hillmyer, M. A.;Bates, F. S. 1996, 9297, 6994-7002 with some modifications. The anionicpolymerization of butadiene was carried out in THE at −60 to −50° C.using sec-butyl-butyllithium as the initiator. A dry 2 neck flask wasdried in the oven overnight and a line was attached to one port with aseptum to another. The flask was flame dried and a stir bar was added.30 ml of Dry Solv THE was added to the 2 neck flask using a cannula. 11ml butadiene (0.13 mol) was condensed in a condensing flask. Liquidnitrogen was first used to condense polybutadiene and then melted usinga dry ice-acetone bath. This was transferred to the 2 neck flask using acannula. 7 ml (0.0098 moles) of 1.4 M sec-butyl lithium initiator wasswiftly added. The polymerization was allowed to proceed for 3 h. Endcapping was accomplished by adding 2 ml (0.051 moles) of ethylene oxideat −60° C. upon complete conversion of the butadiene. Acidic methanol (5ml HCl: 50 ml methanol) was then used to liberate the polybutadienealcohol which was isolated by evaporation of the solvent. Inorganicsalts were removed by extraction of a cyclohexane solution of thepolymer with distilled water. Polymer was left on high vacuum to removewater. Further drying was achieved by refluxing the polymer in dryhexane using molecular sieves in soxhlet extractor.

Step (b): PB-PMOXA Synthesis

20 g (0.0260M) of polybutadiene (Mn 769 g/mol) were functionalized with7.33 g (0.0260M) triflic acid anhydride (SigmaAldrich 176176-5G) in thepresence of 2.63 g (0.0260M) of triethylamine (SigmaAldrich T0886) at−10 deg C. under argon. Organic salts were further filtered out.Triflate-functionalized PB served as a macro-initiator of cationic ringopening polymerization of 2-methyl-2-oxazoline (SigmaAldrich 137448).Polymerisation was allowed to proceed in anhydrous ethyl acetate(SigmaAldrich 270989) at 40 deg C. for 12 h. Reaction was terminatedwith ethylene diamine 0.4 g (SigmaAldrich 03550). This provided primary-and secondary-amine terminated PB-PMOXA polymer.

Polymer Characterization: PB₁₂—OH NMR

5.45 ppm —CH═CH₂ (repeating unit), 4.94 ppm —CH═CH₂ (repeating unit),2.12 ppm CH (repeating unit—backbone), 1.27 ppm CH₂ (repeatingunit—backbone), CH₂ and CH₃ 3.65 ppm 0.82 ppm—end groups.

Polymer Solvent Mn Mw PDI PB₁₂ CHCl₃ 526 602 1.14 PB₁₂PMOXA₅ CHCl₃ 632738 1.19PB₁₂-PMOXA₅-NH—(CH₂)—NH₂

NMR

PB: 5.45 ppm —CH═CH₂ (repeating unit), 4.94 ppm —CH═CH₂ (repeatingunit), 2.12 ppm CH (repeating unit—backbone), 1.27 ppm CH₂ (repeatingunit—backbone), CH₂ and CH₃ 3.65 ppm 0.82 ppm—end groups. PMOXA: 3.45ppm (—CH₂—CH₂—N—), 2.11 ppm (—N—CO—CH₃)

Step (c) Vesicle Preparation

PB₁₂-PMOXA₅-NH—(CH₂)₂—NH₂ polymer (50 mg) was dissolved in 1 mlchloroform in a round bottom flask (Pyrex 200 ml). Solvent wasevaporated on a rotary evaporator under reduced pressure producing athin film of polymer. Subsequent 3 h high vacuum treatment removed thetraces of chloroform. 5 ml of water was further added and stirred at 600rpm. This way a 10 mg/ml suspension of vesicles was prepared. Uponsampling for characterization (LSM, Stopped-Flow, DLS), the suspensionwas extruded successively through polycarbonate Track ached filters(Millipore) of 1 μm, 800 nm, 400 nm, 200 nm. At each of the extrusions,the suspension was sampled for characterization.

The vesicles were characterised as follows. Cryogenic transmissionelectron microscopy (cryo-TEM) was used for particle imaging, andsurface functionalization was studied using LSM imaging.

For the cryo-TEM, the microscope was FEI TecnaiG2, TF20. Samples werevitrified using a vitrification robot, Vitrobot™ FEI. Magnification usedwas 25000× (calibrated 31625×)=scale bar 200 μm.

For the LSM imaging, the amine end groups present on the surface of thevesicles prepared as above were allowed to react withtetramethylrhodamine isothiocyanate fluorescent dye (1:1000 molar ratio)and dialyzed against deionized water. Dialysis was performed untildialysate showed no signs of fluorescence, followed by additional changeof DI water to eliminate unspecific binding. The vesicles werevisualized using a Zeiss LSM 710 Inverted Confocal Microscope withApochromat 63x/1.4 Oil DIC M27 objective and 561 nm Laser line. Pinholewas varied from 50 um to 70 um. This allowed for the confocal plane to“see through” the vesicles, which thus appear as rims of light (centerof vesicle in the center of confocal point) or discs of light (top ofthe vesicle in confocal point) in suspension where a vesicle floated inand out of focus dynamically. FIGS. 7A and 7B show two samplemicrographs clearly showing vesicles.

Step (d): Insertion of Protein into Vesicles

Water permeability of polymer vesicles was enhanced by reconstitution ofwater channel membrane protein—aquaporin Z. Film hydration procedure wasmodified to accommodate addition of protein at PoPr 400. Shortly: to thehydrating vesicles protein solution is added at PoPr 400. Next stepsfollow the protocol of standard vesicles formation.

PB₁₂-PMOXA₅-NH—(CH₂)₂—NH₂ polymer (50 mg) was dissolved in 1 mlchloroform in a round bottom flask (Pyrex 200 ml). Solvent wasevaporated on a rotary evaporator under reduced pressure producing athin film of polymer. Subsequent 3 h high vacuum treatment removed thetraces of chloroform. 5 ml of 100 mM Na-MOPS buffer containing 0.1245 mgof aquaporin Z (Applied Biomimetic) and 0.5% octyl glucoside(O311—n-Octyl-β-D-Glucopyranoside, Anagrade, Anatrace) and was furtheradded and stirred at 600 rpm. 10 mg/ml suspension of proteo-vesicles wasextruded trough 200 nm polycarbonate Track ached filter (Millipore).Permeability measurements were performed using stopped-flowspectrometer.

Stopped flow spectroscopy was used to evaluate protein insertion. Thisis measured as increase in water permeability of vesicles reconstitutedwith aquaporin water channel. With the amount of protein added as littleas PoPR (polymer to protein ratio) of 400 the increase in waterpermeability over control vesicles was measured to be 46 times. Theresults are shown in FIG. 8 .

Step (e): Membrane Preparation

In this Example, the concentration of deposited vesicles was keptconstant and monitored by matching the count rate (250 kcps) in DynamicLight Scattering (Malvern Zetasizer Nano) with static attenuator.

Sulfo-SANPAH (SS) solution (10 mM in 100 mM NaMOPS pH 7.5) was allowedto react with previously prepared PB-PMOXA-NH—(CH₂)₂—NH₂ vesicles in theabsence of light (250 μL of vesicle solution combined with 50 μL SS for15-minutes). A series of 47 mm polysulfone membranes (hand casted) werecut by punch press and placed into Teflon® membrane holders and rinsedwith deionized water. Excess water was removed by compressed air and 300μL (each) of SS-activated vesicle suspensions were placed onto thepolysulfone support membranes. The membrane holders were then placedunder UV light approximately 5 cm from the source and covered with foilfor protection for 30 minutes. Excess reactants were then removed fromthe membrane surface using a 1 ml pipette without touching the membranesurface. The above steps were repeated three times, following which themembranes were removed from the holders and 25 mm diameter membranesamples were cut from the coated area using a punch press. These werethen rinsed in excess 100 mM NaMOPS ph7.5 on a shake table for at leastone hour before testing.

FIG. 9 is a micrograph of the resulting membrane, showing a coherentmass comprising a plurality of vesicles cross-linked on the surface ofthe support membrane.

Membranes prepared in the step described above were subject to treatmentwith either 10 or 150 μL of free radical initiating solution composingof:

-   -   25 mM Iron(II) Sulfate Heptahydrate,    -   25 mM Sodium Metabisulfite,    -   25 mM Potassium Persulfate

The treatment resulted in crosslinking of the PB hydrophobic core.

The resulting membrane samples were tested for pore size distributionusing a standard molecular weight cut-off analysis technique. The 25 mmsamples prepared in the previous step were tested for their ability toretain high molecular weight materials, by measuring their molecularweight cut-off, i.e. the point at which at least 90% of molecules of agiven molecular weight are retained by the membrane. Phosphate buffer(0.03M Na₂HPO₄+0.03M KH₂PO₄) was pre-filtered using a 0.2 um membraneand the pH was adjusted to 7.2 prior to use for preparation ofsolutions. Dextran (DXT) standards were dissolved in phosphate buffer(DXT 165 kDa, 325 kDa, 548 kDa, 1300 kDa, and 5000 kDa, DXT 0.505 kDa, 4kDa, 6 kDa, 11 kDa, 20 kDa, and 28 kDa). All of the dextran solutionswere diluted to 0.5 mg/ml with phosphate buffer and pre-filtrated usinga 0.2 um polyethersulfone membrane prior to use. All filtrationexperiments were conducted in a 10 ml Amicon stirred ultrafiltrationcell (Model 8010, Millipore Corp.) All samples were evaluated accordingto the protocol described below:

-   -   Filtered 10 ml volume of deionised water at 20 psi to wet the        pore structure and the whole system.    -   Connected the feed line with dextran solution feed to a digital        peristaltic pump (Thermal Fisher Science Inc.), re-pressurized        the cell to 20 psi, set the filtrate flux to 5 μm/s.    -   Obtained 800 μL samples of the filtrate solution after        filtration of 2,000 μL of water for equilibration and washing        out the dead volume downstream of the membrane.    -   Obtained 1 ml permeate samples directly from the cell after        filtration.    -   Cleaned and rinsed the whole system with deionised water.    -   The stirring speed was kept at 600 rpm and all experiments were        performed at room temperature (22±3° C.)

Permeate was further evaluated using high-pressure liquid chromatography(HPLC columns PL1149-6840, MW 10,000 to 200,000, PL1120-6830, MW 100 to30,000, PL1149-6860, MW 200,000 to >10,000,000). Comparison of the feedto the permeate chromatograms allowed for calculation of retentioncoefficients and membrane molecular cut-off. The results are shown inFIG. 10 , which shows that molecular cut-off of the control membrane wasreduced to half when coated with vesicles. Molecular weight cut-off ofthe vesicle-coated membrane decreased to 4000 Ka upon core-crosslinkingof the polybutadiene using initiator. Reduction in molecular cut-off isshown to be dependent on the amount of the cross-linker used.

What is claimed is:
 1. A process for the preparation of a filtrationmembrane, which comprises providing an aqueous suspension of vesicleshaving transmembrane proteins incorporated therein, said vesicles beingformed from an amphiphilic block copolymer having reactive end groups;providing a porous support; functionalizing a surface of said poroussupport to introduce reactive groups on said surface which are capableof reacting with the reactive end groups of the amphiphilic blockcopolymers of the vesicles; depositing said suspension of vesicles on asurface of the porous support; and providing reaction conditions suchthat covalent bonds are formed between said vesicles and said surface.2. A process as claimed in claim 1, wherein the reactive groupsintroduced on said surface are selected from the group consisting ofamine groups, carboxylic acid groups, activated carboxylic acid groupsand click chemistry groups.
 3. A process as claimed in claim 1, whereinthe reactive end groups of the amphiphilic block copolymers are selectedfrom the group consisting of amine groups, carboxylic acid groups,activated carboxylic acid groups and click chemistry groups.
 4. Aprocess as claimed in claim 1, wherein the reactive groups introduced onsaid surface and the reactive end groups of the amphiphilic blockcopolymers are respectively: (i) amine and carboxylic acid groups; (ii)amine and activated carboxylic acid groups; (iii) carboxylic acid groupsand amine groups or (iv) activated carboxylic groups and amine groups;or wherein the reactive groups introduced on said surface and thereactive end groups of the amphiphilic block copolymers are clickchemistry groups.
 5. A process as claimed in claim 1, wherein thereactive groups introduced on said surface and the reactive end groupsof the amphiphilic block copolymers are respectively: (i) azide andalkyne groups or (ii) alkyne and azide groups.
 6. A process as claimedin claim 1, wherein said step of functionalizing a surface of saidsupport comprises introducing carboxylic acid groups on said surface. 7.A process as claimed in claim 2, wherein the activated carboxylic acidgroup is an activated ester, e.g. an N-hydroxysuccinimide ester, or anacid halide.
 8. A process as claimed in claim 3, wherein the activatedcarboxylic acid group is an activated ester, e.g. anN-hydroxysuccinimide ester, or an acid halide.
 9. A process as claimedin claim 1, wherein said functionalisation of said surface of saidporous support is achieved via the addition of additives to a castingdope used to form said porous support.
 10. A process as claimed in claim1, wherein said functionalisation of said surface of said porous supportis achieved via chemical treatment, graft polymerization or plasmapolymerization.
 11. A process as claimed in claim 1, wherein saidfunctionalisation of said surface of said porous support is achievedvia: conversion of amine groups on said surface into carboxylic acidgroups, or vice versa; conversion of aldehydes on said surface intoamines; or conversion of hydroxyl groups on said surface into carboxylicacid groups.
 12. A process as claimed in claim 1, wherein said poroussupport comprises polysulfone, poly(ether sulfone), polycarbonate,poly(ether imide), poly(2,6-dimethyl-1,4-phenylene oxide), polyimide,poly(vinylidene fluoride), polytetrafluoroethylene, polypropylene,polyacrylonitrile, poly(methyl methacrylate, polyvinyl alcohol, andpolydimethylsiloxane, regenerated cellulose, cellulose nitrate,cellulose acetate or polyamide.
 13. A process as claimed in claim 1,wherein the support comprises a polyolefin, polyethersulfone,polysulfone or polyacrylonitrile.
 14. A process as claimed in claim 1,wherein the surface of the support is covered with a continuous layer ofvesicles.
 15. A process as claimed in claim 1, wherein the vesicles forma coherent mass on the surface of the support.
 16. A process as claimedin claim 1, wherein the amphiphilic block copolymer comprises at leastone hydrophilic block comprising (poly)₂-C₁₋₃alkyl-2-oxazoline, and atleast one hydrophobic block comprising (poly)dimethyl siloxane orpolybutadiene.
 17. A process as claimed in claim 1, wherein theamphiphilic block copolymer is((poly)₂-C₁₋₃alkyl-2-oxazoline)a-((poly)dimethylsiloxane)b-((poly)₂-C₁₋₃alkyl-2-oxazoline)a in which each aindependently is a number between 5 and 100, and b is a number between 5and
 150. 18. A process as claimed in claim 1, wherein the transmembraneprotein is an aquaporin.